Health promoting drink

ABSTRACT

The present invention relates a new drink formula comprising fresh marine omega-3 oil in an emulsion and antioxidants well known to be health promoting to humans, and process for the production of said drink and the use of said drink for production of a medicament.

FIELD OF INVENTION

The present invention relates a new drink formula comprising freshmarine omega-3 oil in an emulsion and antioxidants well known to behealth promoting to humans, a process for the production of said drinkand the use of said drink for production of a medicament.

DESCRIPTION OF PRIOR ART

The health promoting effects of polyunsaturated oils are well known. Thehealth promoting effects of antioxidants are also known. However, it isa need for a composition combining these nutrients in a formula thatresult in increased absorption and where the body is able to utilize thenutrients optimally.

There is a need for new compositions where these native unstable healthpromoting nutrients are formulated such as each component is keep intactand fresh.

The effects of omega-3 fatty acids (EPA, DHA and DPA) on a number ofdiseases and conditions, such as cardiovascular, mental, skin andageing, are well documented. Supplementation of omega-3 increases worldwide. There is an increase in the consumption of omega-3 containingproducts, and omega-3 in the form of fish and/or food supplement ishighly recommended by health authorities.

Oxidative stress is a sort of “chemical stress” induced by the presencein our body of abnormal quantities of free radicals. Whatever the cause,oxidative stress is believed to be responsible of early ageing and of avery long series of common diseases—about one hundred—that span fromarterial hypertension to atherosclerosis, from infarct to ictus, fromParkinson's to Alzheimer's, from colitis to pancreatitis, from obesityto diabetes, from chronic bronchitis to rheumatoid arthritis, from AIDSto several types of cancer.

The body is protected against free radicals by antioxidants, bothself-produced and antioxidants supplied through food and drinks.Antioxidants may be vitamins, minerals, and enzymes, either fat solubleor water soluble.

In situations where the body is subjected to enhanced oxidation (a lotof free radicals), the body might not have sufficient antioxidants toneutralize or quench the free radicals. Destructive chain reactionsoccur, which might cause increased and detrimental oxidative stress.

A conventional method for measuring the oxidative status is FRAS (FreeRadical Analytical System.) The test is evaluated in a conventionalmeasuring unit called U.Carr (from the chemist Carratelli, the inventorof the test). The method is quick and only a drop of blood drawn fromthe finger tip is required.

The below table gives an overview of normal and elevated oxidativevalues.

FRAS Values:

Normal values 250-300 U.Carr Boarderline values 300-320 U.Carr (showinga slightly elevated level of free radicals which could lead to oxidativestress conditions) Mild oxidative stress 320-340 U.Carr Moderateoxidative stress 340-400 U.Carr Severe oxidative stress 400-500 U.Carr

The theoretical basis for the effects of antioxidants is wellacknowledged. It is also acknowledged that the absorption ofantioxidants in the body from antioxidant supplements is a challenge.However, studies have demonstrated that antioxidants in a non-nativeform or as isolated vitamins are inadequately taken up by the body. Somestudies indicate that ingestion of high dosages of isolated vitamins mayconvert antioxidants to prooxidants, thus leading to elevated oxidationin the body. Studies and literature indicates better absorption andbioavailability of antioxidants naturally present when consumed in foodse.g. as fruits and vegetables.

It is known that humans having severe oxidative stress are oftendeficient in omega-3 fatty acids (DHA and EPA), and possess a lowantioxidative status.

Oxidative damage and antioxidant deficiency is now regarded as crucialfactors to many diseases, and are probably the primary reason for animperfect replacement of old damaged cells by new cells.

Research work has demonstrated that oxidation products of fatty acidsare highly reactive and may affect and interfere with intracellularprocesses. Many commercially available omega-3 supplements contain fishoil having a significant degree of oxidation, which in turn may inducesadverse effects on intracellular processes.

Although these dietary supplements often are added antioxidants, thiswill not reverse the rancidity already present in the dietarysupplement. On the other hand, to prevent further oxidation of theunsaturated fish oil, the antioxidants in the supplement will beconsumed and finally (after some months) cease. Thus, antioxidants inthe commercially available dietary supplements will not induce anyhealth promoting effects in humans.

Thus, there is a need for a new composition combining fresh fish oil andspecific antioxidants to provide a new drink formula having improvedhealth promoting effects on humans.

PRESENT INVENTION

Thus, the object of the present invention is to provide a new drinkformula comprising fresh marine omega-3 oil and antioxidants well knownto be health promoting to humans.

A further object of the present invention is to provide a process forthe production of the new drink formula, wherein the fish oil isemulsified and handled under gentle conditions through a minimum ofprocess steps.

A further object of the present invention is to provide a drink for useas a therapeutic drink. The specific antioxidants to be included in thedrink are selected according to the disease or disorder to be treated.

These and further objects are achieved by the present invention.

Hence, the present invention relates to a drink combining fresh marineoil and specific antioxidants. The antioxidants having a healthpromoting effect on humans are present in the drink to reduce oxidativestress of the human being, not for the purpose of oil stabilization inthe preparation. The drink may be a functional drink or a therapeuticdrink, a thirst-quencher or an ordinary drink. Preferably, the drink hasa base containing natural antioxidants eg. fruit or vegetable juice,green tea, but any drinkable liquid may be used.

The new drink combines the stable omega-3 emulsion known from prior art,and well known health promoting antioxidants. The drink revealssurprising characteristics as to absorption of antioxidants and theeffect of antioxidants on the body. Compared to prior art wherecorresponding amounts of omega-3 and antioxidants are ingested indifferent formulas, the drink according to the present invention showsimproved delivery, improved uptake and improved effect on oxidativestress.

Both the omega-3 oil and the antioxidants contained in the drinkaccording to the invention are remarkable stable in the composition, andthe progress of rancidity and loss of antioxidant effects are much lowerthan in known product formulated as separate capsules.

One aspect of the present invention relates to a drink comprising freshmarine oil in an oil-in-water emulsion wherein the marine oil has atotox value below 10, further added at least one antioxidant notnaturally present in said oil-in-water emulsion.

The term fresh marine oil describes an oil prepared from fresh fishwhere all process steps are to conducted carefully and under strictoxygen control according to functional oil standards in order to preventoil oxidation. The fresh marine oil will have a low oxidative status,revealing a colourless oil without the characteristic smell or taste offish. The level of oxidation given as the totox value (2 times theperoxide value (PV) added with the anisidin value (AV)) should be below10, preferably below 8, and most preferably below 5. Marine oil presentin many food supplements today contains oil with a much higher totoxvalue, typically 20-30 or even higher.

The fresh marine oil may be any oil rich in omega-3, e.g. fish oil, sealoil or krill oil. The oil may be mixed with other polyunsaturated oilsof vegetable origin such as algae oil and herbal oil such as eveningprimrose oil and rapeseed oil.

One preferred embodiment of the present invention provides a drinkwherein the content of marine oil is about 0.5%-5% by weight based onthe total weight, more preferably in the range of 0.5%-3%, mostpreferably in the range of 1.5% 2.5%.

The oil-in water emulsion is prepared by any conventional method,preferably as described in the applicants own Norwegian applications NO20044542, 20053136 and 20055620. In said emulsions the antioxidants arepresent to stabilize the oil during production and storage, not for thepurpose of inducing any health promoting effects on humans.

The water phase of the oil-in water emulsion is preferably a water phasecontaining natural antioxidants e.g. fruit/vegetable juices, green tea,white tea and herbal tea. The juice may be a fresh pressed juice orjuice in the form of juice concentrate or juice purrè diluted to obtaina normal ready-to-use juice. The water phase may also contain proteinssuch as soy, oat proteins, whey proteins and/or milk proteins.

According to the invention, the term added or further added antioxidantsare to be understood as antioxidants not naturally present in the wateror oil phase, but added separately. The further added antioxidant may besimilar to one or more antioxidants naturally present in the water oroil phase, or different. The further added at least one antioxidant maybe one or a combination of two or more antioxidants.

Antioxidants useful according to the present invention are antioxidantsknown to have health promoting effects on humans. Useful antioxidantsare e.g. astaxanthin, vitamin E, vitamin C, beta-carotene, lutein,lycopene, zeaxanthine, glutathione, flavonoids, carotenoids, to plantphenols, polyphenols, coenzym Q10, reservatrol, curcumin, pycnogenol,selen, copper, zink and magnesium. The antioxidants may be presentindividually or in a mixture of two or more. The antioxidants may beselected to meet specific purposes, i.e. the desirable health effects tobe achieved. As examples, a beverage according to the inventioncomprising lutein and zeaxanthine will likely have a positive impact ondifferent eye conditions. A beverage is according to the inventioncomprising the antioxidant rich curcumin is suitable to alleviateinflammation and fight infections. A beverage according to the inventioncomprising CoQ10 may be useful for neurological disorders. Further, abeverage according to the invention comprising pycnogenol may bebeneficial for reducing osteoarthritis. Beta-carotene added to the saidbeverage may be useful for respiratory disorders and CoQ10 and seleniumwill be useful for cardiovascular disorders.

In one embodiment of the present invention, the drink may be addedprebiotics and/or probiotics.

In one embodiment of the present invention, the drink may be carbonated.

A further aspect of the present invention relates to a process for theproduction of a drink according to the invention comprising thefollowing steps:

a) oil soluble antioxidants and flavouring agents, together withemulsifier are added to the oil phase,b) water soluble additives are added to the water phase,c) the oil and water phase are mixed to a homogenous emulsion,d) the emulsion obtained is optionally subjected to pasteurizationand/or homogenization processes,e) the obtained emulsion is cooled down and filled on clean disposablecontainers;wherein all steps are performed under strict oxygen control.

Alternatively, the process for the production of a drink according tothe invention comprises the following steps:

a) oil soluble antioxidants and flavouring agents are added to the oilphase,b) water soluble additives are added to the water phase,c) the oil and water phase are mixed and the emulsifier is added,followed by gentle mixing to achieve a homogenous emulsion,d) the emulsion obtained is optionally subjected to pasteurizationand/or homogenization processes,e) the obtained emulsion is cooled down and filled on clean disposablecontainers;wherein all steps are performed under strict oxygen control.

A further aspect of the present invention relates a drink for use as atherapeutic drink. Further aspects relate to the use of a drinkaccording to the invention for the production of a medical preparationfor the prophylaxis or treatment of diseases associated with elevatedoxidative stress, such as cancer, inflammatory disorders, neurologicaldisorders, cardiovascular disorders and respiratory disorders.

It has surprisingly been found that the new drink formula combiningfresh omega-3 fatty acids and at least one added antioxidant have animproved health promoting effect compared to food supplements wheremarine oil and antioxidants are ingested in separate formulas, e.g.capsules, pills or tablets. The specific formulation of the invention isbelieved to be of great importance, presenting the essential nutrientsand specific health promoting agents (polyunsaturated fatty acids andadded antioxidants) to the digestive system and to the cells in a formathighly beneficial to the cells and the body.

FIGURES

FIG. 1 shows the effect of marine oils on lipid peroxidation in U937cells. Cells were incubated for 48 h with different concentrations ofomega-3 fatty acids from different vendors as shown in legend.Concentrations are indicated in the X-axis. Y-axis shows the relativelevels of lipid peroxidation using DPPP mediated fluorescence as asensor. Standard deviation is indicated.

EMBODIMENTS

The invention will now be further illustrated with reference to thefollowing non-limiting examples.

Drink According to the Invention

The marine oil used in all preparations was Xalar salmon oil from MarineHarvest Ingredients, batch number: 099000F016. The totox value was below5 and the preparation were conducted according to functional oilstandards.

Example 1

Drink comprising fresh marine omega-3 emulsion and astaxanthin.

% Water, purified 42.31 Vegetable juice 45.00 Rosemary Extract 201 0.02Toco 50 0.01 Grindsted 3115 1.00 Apple juice concentrate 10.00 Nat lemonaroma 0.15 Astaxanthin 0.01 Marine oil 1.50 sum 100.00Granini Vegetable juiceGrindsted 3115 from DansicoNat lemon aroma from FirmencihFood grade Haematococcus pluvialis powder (esterified astaxanthincomplex in the form of haematococcus pluvialis algae biomass), suppliedby: Sinochem Hebei corporation, 707 Lianmeng, Shijiazhuang, China

Example 2

Drink comprising fresh marine omega-3 emulsion supplied with vitamin Cand E.

% kg Rosemary Extract 201 0.02 0.16 Toco 50 0.01 0.08 Grindsted 31151.00 8.00 Apple concentrate 6.05 48.40 Pomegranate apple concentrate2.40 19.20 Aronia concentrate 0.80 6.40 Pear concentrate 6.49 51.92Water, purified 81.00 648.00 Orange/mandarine Flavour 0.10 0.80Raspberry Flavour 0.12 0.96 Vitamin C 0.01 0.80 Selenium 0.4 Vitamin E0.08 Marine oil 2.00 16.00 Sum 100.00 800.80Natural Raspberry Flavour from FirmenichNatural Orange/Mandarin Flavour from FirmenichVitamin E: D-alfa, D-beta, D-delta, D-gamma tocopherol and D-gammatocotrenolVitamin C: Calsium-L-ascorbate (acid neutral)Selenium: L(+)selenium methionine;all from G.O Johnsen

Example 3

Drink comprising emulsified fresh marine omega-3 fatty acids andlycopene and lutein.

% Rosemary extract 201 0.020 Toco 50 0.01 Grindsted 3115 1.00 Wheyprotein 0.45 Apple concentrate 6.23 Pommegranat concentrate 2.40 Aroniaconcentrate 0.88 Pear concentrate 5.56 Water, purified 81.48Orange/mandarine flavour 0.10 Raspberry flavour 0.20 Marine oil 1.50Lyconat (6% lycopene) 0.12 Lutenat (10% lutein) 0.05 sum 100.00Rosemary extract 201, Toco 50 and Grindsted 3115 from DansicoWhey protein from Arla FoodsNatural flavouring agents from FirmenichNatural lycopene (Lyconat) from VitateneNatural lutein (Lutenat) from Vitatene

Example 4

Drink comprising emulsified fresh marine omega-3 fatty acids and lutein.

% Rosemary extract 201 0.020 Toco 50 0.01 Grindsted 3115 1.00 Appleconcentrate 3.23 Pommegranat conc 1.40 White grape concentrate 2.56 Soyamilk 58.40 Water, purified 31.42 Apricot flavour 0.25 Lemon flavour 0.10Marine oil 1.50 Lutein 0.11 sum 100.00Rosemary extract 201, Toco 50 and Grindsted 3115 from DansicoNatural flavouring agents from FirmenichSalmon oil for Marine Harvest Ingredients, Totox below 5 and processedaccording to functional oil standardsNatural lutein (Lutenat 10% OS) from Vitatene, Italy

Example 5

Drink comprising emulsified fresh marine omega-3 fatty acids and CoQ10and selenium.

% Rosemary extract 201 0.02 Toco 50 0.01 Whey proteins 0.35 Grindsted3115 1.20 Apple concentrate 6.66 Pommegranat concentrate 1.90 Aroniaconcentrate 2.56 Water, purified 84.15  Mangosteen flavouring 0.25 Umbuflavouring 0.10 Marine oil 2.50 CoQ10 (10%) 0.30 Selenium  50 μg VitaminD 1.5 μg sum 100.00 Rosemary extract 201, Toco 50 and Grindsted 3115 from DansicoNatural flavouring agents from CargillSelenium supplied by: Sinochem Hebei corporation, 707 Lianmeng,Shijiazhuang, ChinaCoenzyme Q10 from DSMVitamine D from G.O JohnsenWhey proteins (Lacprodan) from Arla Foods

Example 6 Process for Production 1. Oil Phase

The oil is mixed with rosemary extract, Toco 50 (an antioxidantpreparation favourable to the is stabilization of the oil). It isimportant that the oil is protected against oxidation during processing.Thereafter, the emulsifier is added (Grindsted 3115) to the oil, mixedgently at room temperature to a homogenous mixture. The flavouringagents are then added.

2. Antioxidants

Oil soluble antioxidants are mixed with the oil phase, and the watersoluble antioxidants are mixed with the water phase prior to heating.Optionally, the water soluble antioxidants are mixed with the waterphase after heating through specialized systems such as Flexdose (TetraPack solution), or added into a sterile tank through an ultra pureprocess.

3. Water Phase

A tank is filled with purified and deionised water. The oil phase isadded to the water phase.

Optionally, the oil phase may be mixed with the water phase after whichthe emulsifier is added and an emulsion is obtained. The fruitconcentrates are then added to the emulsion obtained and mixedthoroughly.

The obtained emulsion may alternatively be subjected to a quickpasteurization (about 90° C. for 8 s), followed by homogenization andcooling to a temperature of 4-8° C.

Finally, the drink is filled on airtight aseptic containers, preferablysingle dose containers, e.g is Tetra Brick about 200 ml and stored at6-8° C. until use.

Strict oxygen control must be implemented in all steps to avoidoxidation of marine oil.

Health Promoting Effects of the Drink According to the InventionMaterials and Methods

To investigate the physiological effects of the drink according to theinvention, oxidation of PUFAs in cell membranes has been analyzed. Afluorescent sensor of lipid peroxidation nameddiphenyl-1-pyrenylphosphine (DPPP) was used to assess lipidperoxidation. DPPP incorporates rapidly into the cell membrane and uponoxidation emits light, which can be measured by a fluorometer (Okimotoaet al., FEBS Letter, June 2000). Samples according to the invention andsamples containing fish oil from commercial available marine oilcapsules were analyzed and compared.

Sample Preparation

Drink as described in Example 3 were used and identified as sample1=Smartfish.

The drink contains 900 mg omega-3 pr 200 ml of drink. The amount oflycopene and lutein are 16 mg and 10 mg lutein pr 900 mg omega-3,respectively.

Best before: 15 Aug. 2009. Fresh fruity taste and smell. No smell ortaste of fish oil could be experienced. Homogenous dark red juicyappearance.

Reference samples were prepared from commercial available capsulescontaining marine oil.

Reference sample 1=TriomegaTriomega (Lot no: 56416408; Midelfart) containing 620 mg omega-3 pr goil.Best before: 6 Nov. 2010. Color: Clear. Odour: Weak but detectable.Reference sample 2=Omega 3Omega 3 (Lot no: 58212708, Eldorado) containing 570 mg omega-3 pr g oil.Best before: 27 Oct. 2010. Color: clear; Odour: weak but detectableReference sample 3=Direct CareCAREMAX (Lot no 71105, Direct Care) containing 600 mg omega-3 pr g oil.Best-before: October 2009; Color: brown; Odour: strongReference sample 1 and 2 is available in the shops and were bought atthe grocery store. Reference sample 3 was ordered via internet.

Marine oils were collected from the capsules with a syringe and mixedwith the same emulsifier as used in the drink according to theinvention, i.e. Grindsted 3115. 1.5 g of marine oil was mixed with 0.75g emulsifier (marine oil:emulsifier 2:1). Cell medium was added to givea total volume of 10 ml to yield an oil-in-water emulsion. Addition ofmedium was followed by vortexing.

Sample 1 is added lycopene and lutein. Thus, the marine oils fromcapsules were supplemented with lycopene and lutein to correspond to theamount of lycopene and lutein present in sample 1.

Lycopene and lutein were purchased from Sigma-Aldrich and dissolved inDMSO (Sigma-Aldrich) to stock solutions containing 3.6 μg/μl or 2.2μg/μl, respectively. 10 μl of the stock solutions were added to 316 μlmarine oil of the reference samples to achieve corresponding amounts oflycopene and lutein as in sample 1 of the invention. Table 1 below showsthe preparation of stock solutions and addition to the referencesamples.

TABLE 1 Preparation of stock solutions and addition of lycopene andlutein Vol added to Lycopene in DMSO Lutein in DMSO 3160 μM ω3 Stocks3.6 μg/μl 2.2 μg/μl 10 μl of each (1 mg/277 μl DMSO) (1 mg/454 μl DMSO)

TABLE 2 Overview of sample preparation Omega-3 (ω3) Mol ω3/g LycopeneLutein Sample 1 900 mg/200 ml juice   14 μmol/g juice   16 mg/900 mg ω3  10 mg/900 mg ω3 Smartfish Ref. sample 1 620 mg/g oil 1.94 mmol/g oil  11 mg/620 mg ω3  6.8 mg/620 mg ω3 Triomega Ref. sample 2 570 mg/g oil1.78 mmol/g oil 10.1 mg/570 mg ω3 10.1 mg/570 mg ω3 Omega 3 Ref sample 3600 mg/g oil 1.87 mmol/g oil 10.7 mg/600 mg ω3 10.7 mg/600 mg ω3 Directcare For all   18 μg/mg ω3   11 μg/mg ω3 samples

Cells

U937 (ATCC No CRL-1593.2™), a human monoblast cell line, which arestably transfected with NF-κB-RE coupled with luciferase. Cells weregrown in RPMI+L-glut, 10% FCS, 2×P/S, Hygromycin.

Cells were incubated with sample 1 and reference sample 1-3 inconcentrations of omega-3 fatty acids ranging from 1 μM to ˜3000 μM for48 hours. The samples were supplemented with lipoprotein lipase, whichhydrolyzes and liberates fatty acids from the glycerol backbone of thetriglycerides as described below.

Probe for Lipid Peroxidation:

Dipenyl-1-pyrenylphosphine (DPPP, D7894, lot no 29055W) was purchasedfrom Invitrogen. MW 386.43 g/mol, 5 mg. Excitation/emission: 351/380 nm.Stock solution: 5 mM (5 mg in 2.6 ml DMSO).

Lipase

10 Units of bovine milk lipoprotein lipase (from Sigma-Aldrich, L2254,lot no 114K7430) was added to the cells containing the highestconcentrations of omega-3 fatty acids in the marine oils. Followingdilution of the samples, the lipase was diluted accordingly. Thus thelowest concentration of the omega-3 rich samples (1 μM) contained 0.003Units of lipoprotein lipase.

Treatment of Cells and Measurements of DPPP Oxidation, LDH and ProteinContent

Sample 1 according to the invention and reference samples 1-3 containinglycopene and lutein were prepared as stock solutions containing 14 000μM and 300 000 μM of omega-3 fatty acids, respectively. Theconcentrations of omega-3 fatty acids were calculated on the basis ofthe omega-3 content listed in the product descriptions. Since thesamples are fish oils, the omega-3 fatty acids are presented to cells astriglycerides, and not free fatty acids. They were further diluted togive final concentrations in the cell medium ranging from 1 to 3160μmol/L in triplicates (see table 5 for plate set up). Lycopene, luteinand lipoprotein lipase were added to dilute samples destined for cellswith 3160 μM omega-3, and further diluted according to table 3 and 4.Cells (10⁵) were seeded in 96 well plates with 100 μl medium. Prior toaddition of samples, medium was changed and cells were added 50 μlmedium/well. 50 μl of the different dilutions containing all theindicated ingredients were then added to cells. Cells were thenincubated for 48 h at 37° C. and prepared for assessment of DPPPfluorescence according to a slightly modified method described byOkimotoa (Okimotoa et al., FEBS Letter, June 2000)

TABLE 3 Dilutions of sample 1 according to the invention Conc. (μM) VolVol Medium Total vol Omega-3 (μL) (μL) (μL) Oil 14000  2000 stock 6320*450 550 1000 2000* 316 684 1000  632* 316 684 1000  200* 316 684 1000   63.2* 316 684 1000  20* 316 684 1000     6.32* 316 684 1000   2* 316684 1000

TABLE 4 Dilution of reference samples 1-3 Conc. (μM) Vol Vol MediumTotal vol Omega-3 (μL) (μL) (μL) Oil 300000   stocks 6320* 21 979 10002000* 316 684 1000  632* 316 684 1000  200* 316 684 1000    63.2* 316684 1000  20* 316 684 1000     6.32* 316 684 1000   2* 316 684 1000*Concentrations are indicated as 2x the final conc. in cell mediumbecause it was further diluted when 50 μl of the solutions were added tocells containing 50 μl cell medium.

TABLE 5 Plate setup; Omega-3 concentration DPPP + + + + Lipase + + + +Sample 1 Ref sample 1 Ref sample 2 Ref sample 3 “Smartfish” “Triomega”“Omega 3” “Care direct” 1 2 3 4 5 6 7 8 9 10 11 12 A 3160 μM  3160 μM 3160 μM  3160 μM  B 1000 μM  1000 μM  1000 μM  1000 μM  C 316 μM 316 μM316 μM 316 μM D 100 μM 100 μM 100 μM 100 μM E 31.6 μM  31.6 μM  31.6 μM 31.6 μM  F  10 μM  10 μM  10 μM  10 μM G 3.16 μM  3.16 μM  3.16 μM  3.16μM  H  1 μM  1 μM  1 μM  1 μM For DPPP measurements cells were washed 2times in phosphate buffered saline and added DPPP to a finalconcentration of 50 μmol/L. This was done in dark conditions to avoidlight induced oxidation of DPPP. The cells containing the DPPP were thenmeasured for fluorescence using a spectrofluoremeter (SpectramaxMolecular Devices) 3 hours after addition of DPPP.

Protein measurements were done in cells lysates after DPPP assessments.Proteins were measured according to a Bio-rad kit (colorometricabsorbance assay), and used to calculate the relative values.

Results

Table 6 showing normalized fluorescent values in Smartfish, emulsion(control) and reference samples following incubation of U937 cells for48 h. Fluorescence is based on oxidation of DPPP with subsequent changein fluorescent intensity. The data are presented as normalized valuescorrected for protein content in cell lysates. +/− standard deviation isindicated.

TABLE 6 Ref. Ref. Ref. Omega-3 Sample 1 Control sample 1 sample 2 sample3 concentrations Smartfish Emulsion Triomega Omega 3 Care direct   1 μMDPPP 100.0 100.0 100.0 100.0 100.0 SD ±7.0 ±7.9 ±2.1 ±4.7 ±9.6 3.16 μMDPPP 98.0 100.0 97.7 101.2 101.6 SD ±7.5 ±8.0 ±1.1 ±8.6 ±6.7   10 μMDPPP 97.6 98.4 106.2 106.1 111.9 SD ±6.2 ±3.8 ±5.5 ±13.3 ±4.6 31.6 μMDPPP 102.2 101.8 106.0 117.9 114.9 SD ±9.8 ±2.1 ±9.7 ±17.0 ±8.9  100 μMDPPP 95.1 105.1 98.5 132.1 121.7 SD ±9.9 ±3.7 ±0.8 ±23.5 ±11.8  316 μMDPPP 94.2 113.3 107.2 132.9 137.1 SD ±14.2 ±3.7 ±11.1 ±19.2 ±8.1 1000μM  DPPP 97.2 102.3 110.6 136.3 168.9 SD ±7.6 ±11.5 ±2.7 ±43.6 ±11.73160 μM  DPPP 95.2 98.2 121.9 176.0 239.1 SD ±15.1 ±9.2 ±13.0 ±31.7±12.8

Effect of Marine Oils on Lipid Peroxidation in U937 Cells

The results show that no detectable lipid peroxidation was observed incells incubated with the sample according to the invention at any of thedoses used, when compared with controls receiving cell medium alone(data not shown) or an emulsion with no oil added. For the referencesamples, a dose dependent increase in lipid peroxidation was found.Notably, the degree of lipid peroxidation was coinciding with the odourand color appearance of the marine oil products. Odour and color isindicative of oxidation of the oil. Addition of lycopene and lutein tothe oils in comparable concentrations as in sample 1 according to theinvention was not sufficient to inhibit lipid peroxidation.

As seen in the table 6 and in FIG. 1, no effect in DPPP mediatedfluorescence was observed with the two lowest concentrations, i.e. 1 μMand 3.16 μM, of the sample according to the invention or the referencesamples. This indicates that at these concentrations no detectable lipidperoxidation in the cell membrane has occurred. At higherconcentrations, a weak increase is observed for all the oils, except thesample according to the invention. From 10 μM and upwards, a gradualchange in signal seems to be evident. At 100 μM there is a significantdifference between sample 1 according to the invention and referencesample 3, (Care direct) (p=0.045), while a tendency is noticed forreference sample 1 (Triomega) and reference sample 2 (Omega 3). At 316μM and 1000 μM a significant difference between the sample according tothe invention and all reference samples were found. The difference waseven more pronounced at the highest concentration i.e. 3160 μM Thehighest degree of lipid peroxidation was found with reference sample 3(Care direct), while reference sample 1 (Triomega) gave the lowestdegree of lipid peroxidation of the three commercially available marineoil products. Notably, the sample according to the invention did notaffect lipid peroxidation at any of the concentrations used.

Studies in humans have shown that ingestion of omega-3 fatty acids assupplements can reach 10-20% of total fatty acids in plasma (review byMasson et al., J Cardiovasc. Med, 2007). Since the plasma concentrationsof total fatty acids as triglycerides are in the range of 1-5 mM, fishoil plasma concentrations can reach 100 to 1000 μM following oral intakeof fish oil. In the present study, we found increased lipid peroxidationby all the reference samples in this concentration range, which can beregarded as physiologically relevant.

Surprisingly we have found that Sample 1 according to the inventionoffer a greater protection against lipid peroxidation than the referencesamples. No detectable change in lipid peroxidation was found for any ofthe concentrations used for sample 1. A dose-dependent increase in lipidperoxidation were observed for all tree reference samples, despite thefact that the shelf life of all capsules was not expired and allcapsules contained antioxidants to stabilize the fish oil duringstorage. The effect on lipid peroxidation of capsule based marine oilsdiffered between the suppliers. The strongest odour of fish oil andcolouring of oil (brown), which are indicative of oxidation was found inreference sample 3 (Care direct), which also resulted in more lipidperoxidation. The shelf life of this product expires October 2009.Reference sample 1 and 2 expiring October/November 2010 did also inducesignificant lipid peroxidation of the cells. The addition of lycopeneand lutein to the reference samples was evidently not affording enoughprotection to inhibit lipid peroxidation.

Thus, the present invention provides a new drink formula wherein thefresh fish oil and the is added antioxidants are protecting the cellsagainst oxidative stress in a much more efficient way than commerciallyavailable fish oils added corresponding amounts of antioxidants. Asoxidative stress is believed to be responsible for a long series ofdiseases, this new drink formula has a great potential as a healthpromoting drink.

1. A drink comprising fresh marine oil in an oil-in-water emulsionwherein the marine oil has a totox value below 10, and furthercomprising at least one antioxidant not naturally present in saidoil-in-water emulsion wherein the content of marine oil is about 0.5% to5% by weight based on the total weight of the drink and the at least oneadded antioxidant is curcumine that is not naturally present in thewater or oil phase, but is added separately to the drink.
 2. The drinkaccording to claim 1, wherein the totox value is below
 8. 3. The drinkaccording to claim 2, wherein the totox value is below
 5. 4. (canceled)5. The drink according to claim 1, wherein the content of marine oil isabout 0.5%-3% by weight based on the total weight.
 6. The drinkaccording to claim 5, wherein the content of marine oil is about1.5%-2.5% by weight based on the total weight.
 7. The drink according toclaim 1, wherein the water phase in the oil-in-water emulsion comprisesnatural antioxidants naturally present in the water phase.
 8. The drinkaccording to claim 7, wherein the water phase is a juice base.
 9. Thedrink according to claim 7, wherein the water phase is a tea base. 10.The drink according to claim 1, wherein the water phase furthercomprises proteins.
 11. The drink according to claim 10, wherein theprotein is whey protein. 12.-28. (canceled)